Study of histone H4 acetylation in Trypanosoma cruzi

The Trypanosoma cruzi histones are very distinct from other eukaryotes, mainly in the N-terminus, where post translational modifications occur, which are essential for gene expression regulation and chromatin architecture. Among these modifications we found that lysines 4, 10 and 14 of H4 histone are acetylated (H4K4ac, and H4K10ac H4K14ac). In this thesis we show that the H4K4ac, which is the most abundant modification, is enriched in densely packed chromatin, while H4K10ac and H4K14ac are located in less dense chromatin, preferentially at the interface between euchromatin and heterochromatin. H4K4ac decreases in trypomastigote, which is the infective and non-dividing form of the parasite whileH4K14ac increases during G2 and mitosis of the cell cycle in replicative forms. H4K10ac and H4K14ac increases during DNA repair of double stranded breaks while H4K4ac decreases after DNA damage. TcRad51, an essential protein in the homologous recombination pathway, when overexpressed, increases the H4K10ac and H4K14ac levels. When the lysines 4, 10 and 14 are individually replaced by arginine (H4K4R, H4K10R and H4K14R) to prevent the acetylation, they are still incorporated into chromatin and decrease the specific modification level. The H4K4R location is different from the endogenous H4K4ac and the H4K14R expression causes growth reduction, with cells accumulating in mitosis. Mutants H4K10R and H4K14R also have high mortality rates after ƒ× irradiation that causes DNA double-stranded breaks. We also showed that TcBDF2 protein contains a bromodomain that recognizes preferentially H4K10ac. Although the TcBD2 function is unknown, it is increased after UV light exposure, suggesting its involvement in DNA repair. These data together provide evidence that each H4 acetylation has a distinct role in T. cruzi. Probably K4 is involved in chromatin assembly during replication while K10 and K14 acetylation appears to be involved in chromatin remodelling during DNA repair and maybe DNA transcription.As histonas de Trypanosoma cruzi sao bastante divergentes quando comparadas aos demais eucariotos, principalmente na porcao N-terminal, onde ocorrem modificacoes pos traducionais, que sao reconhecidamente essenciais para o controle da expressao genica e da arquitetura da cromatina. Entre estas modificacoes estao as acetilacoes das lisinas 4, 10 e 14 da histona H4 (H4K4ac, H4K10ac e H4K14ac). Nesta tese mostramos que a H4K4ac, que e a modificacao mais abundante, esta enriquecida em areas de cromatina densamente compactada, enquanto H4K10ac e H4K14ac localizam-se em regioes de cromatina menos densa, preferencialmente na interface entre a eucromatina e a heterocromatina. H4K4ac diminui nas formas nao replicativas e H4K14ac H4K14ac aumenta durante G2 e mitose do ciclo de divisao celular das formas replicativas. H4K10ac e H4K14ac aumentam e H4K4ac diminui no reparo de quebras de dupla fita do DNA. Ao mesmo tempo a superexpressao da proteina TcRad51, essencial para o processo de reparo de DNA por recombinacao homologa causa aumento de H4K10ac e H4K14ac. Quando as lisinas 4, 10 e 14 sao substituidas separadamente por argininas (H4K4R, H4K10R e H4K14R) para evitar a acetilacao sao expressas, elas sao incorporadas na cromatina e diminuem os niveis de cada modificacao. A expressao de H4K4R tem uma distribuicao diferente das histonas endogenas e H4K14R causa diminuicao de crescimento, com celulas acumulando na fase de mitose. Os mutantes de H4K10 e H4K14 ainda apresentam maior mortalidade quando submetidos a radiacao ƒ× que causa quebra de dupla fita de DNA. Tambem mostramos que a proteina TcBDF2, uma proteina que contem bromodomineo, reconhece preferencialmente H4K10ac. A funcao da TcBD2 e desconhecida, mas verificamos que ela aumenta apos exposicao dos parasitas a luz UV, sugerindo que esteja participando no reparo de DNA. Esses dados juntos fornecem evidencias de que cada uma das acetilacoes da histona H4 tem um papel distinto no T. cruzi. A acetilacao em K4 estaria envolvida na montagem da xiii cromatina na fase de replicacao. Ja as modificacoes em K10 e K14 estariam envolvidas com processos especificos de remodelagem da cromatina durante os eventos de reparo e eventualmente transcricao do DNA.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)TEDEBV UNIFESP: Teses e dissertaçõe

Identification of an atypical peptidyl-prolyl cis/trans isomerase from trypanosomatids

The parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases) catalyzes the cis/trans isomerization of the peptide bonds preceding Pro residues. Eukaryotic parvulin-type PPIases have been shown to be involved in cell proliferation and cell cycle progression. Here we present the biochemical and molecular characterization of a novel multi-domain parvulin-type PPIase from the human pathogenic Trypanosoma cruzi, annotated as TcPar45. Like most other parvulins, Par45 has an N-terminal extension, but, in contrast to human Pin1, it contains a forkhead-associated domain (FHA) instead of a WW domain at the N-terminal end. Par45 shows a strong preference for a substrate with the basic Arg residue preceding Pro (Suc-Ala-Arg-Pro-Phe-NH-Np: k(cat)/K(M) = 97.1 /M/s), like that found for human Part14. in contrast to human Pin1, but similarly to Par14, Par45 does not accelerate the cis/trans interconversion of acidic substrates containing Glu-Pro bonds. It is preferentially located in the parasite nucleus. Single RNA interference (RNAi)-mediated knock-down showed that there was a growth inhibition in procyclic Trypanosoma brucei cells. These results identify Par45 as a phosphorylation-independent parvulin required for normal cell proliferation in a unicellular eukaryotic cell. (C) 2010 Elsevier B.V. All rights reserved.DAADConsejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET, Argentina)Agencia Nacional de Promocion Cientifica y Tecnologica (ANPCyT, Argentina)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)INGEBI CONICET, Buenos Aires, DF, ArgentinaUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilMax Planck Res Unit Enzymol Prot Folding, Halle, GermanyUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

Chromatin and nuclear organization in Trypanosoma cruzi

A total of 100 years have passed since the discovery of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas' disease. Since its discovery, the molecular and cellular biology of this early divergent eukaryote, as well as its interactions with the mammalian and insect hosts, has progressed substantially. It is now clear that this parasite presents unique mechanisms controlling gene expression, DNA replication, cell cycle and differentiation, generating several morphological forms that are adopted to survive in different hosts, in recent years, the relationship between the chromatin structure and nuclear organization with the unusual transcription, splicing, DNA replication and DNA repair mechanisms have been investigated in T. cruzi. This article reviews the relevant aspects of these mechanisms in relation to chromatin and nuclear organization.Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilInst Butantan, Parasitol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilWeb of Scienc

Trypanosoma cruzi bromodomain factor 2 (BDF2) binds to acetylated histones and is accumulated after UV irradiation

Histone tail post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) regulate many cellular processes. Among these modifications, phosphorylation, methylation and acetylation have already been described in trypanosomatid histones. Bromodomains, together with chromodomains and histone-binding SANT domains, were proposed to be responsible for histone code reading. the Trypanosoma cruzi genome encodes four coding sequences (CDSs) that contain a bromodomain, named TcBDF1-4. Here we show that one of those, TcBDF2, is expressed in discrete regions inside the nucleus of all the parasite life cycle stages and binds H4 and H2A purified histones from T. cruzi. Immunolocalization experiments using both anti-histone H4 acetylated peptides and anti-TcBDF2 antibodies determined that TcBDF2 co-localizes with histone H4 acetylated at lysines K10 and K14. TcDBF2 and K10 acetylated H4 interaction was confirmed by co-immunoprecipitation. It is also shown that TcBDF2 was accumulated after UV irradiation of T. cruzi epimastigotes. These results suggest that TcBDF2 could be taking part in a chromatin remodelling complex in T. cruzi. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier B.V. All rights reserved.National Research Council (CONICET), ArgentinaANPCyTCONICETFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Inst Mol & Cellular Biol, Fac Ciencias Bioquim & Farmaceut, RA-2000 Rosario, Santa Fe, ArgentinaUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Inst Ciencias Biomed, Dept Parasitol, BR-04023062 São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, BR-21949900 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Inst Ciencias Biomed, Dept Parasitol, BR-04023062 São Paulo, BrazilWeb of Scienc

Trypanosoma cruzi XRNA granules colocalise with distinct mRNP granules at the nuclear periphery

BACKGROUND Eukaryotic ribonucleoprotein (RNP) granules are important for the regulation of RNA fate. RNP granules exist in trypanosomatids; however, their roles in controlling gene expression are still not understood. XRNA is a component of granules in Trypanosoma brucei but has not been investigated in Trypanosoma cruzi. OBJECTIVES This study aimed to investigate the TcXRNA dynamic assembly and its interaction with RNP components under conditions that affect the mRNA availability. METHODS We used in vitro metacyclogenesis of T. cruzi to observe changes in RNP granules during the differentiation process. TcXRNA expression was analysed by Western blot and immunofluorescence. Colocalisation assays were performed to investigate the interaction of TcXRNA with other RNP components. FINDINGS TcXRNA is constantly present during metacyclogenesis and is localised in cytoplasmic granules. TcXRNA does not colocalise with TcDHH1 and TcCAF1 granules in the cytoplasm. However, TcXRNA granules colocalise with mRNP granules at the nuclear periphery when mRNA processing is inhibited. MAIN CONCLUSIONS TcXRNA plays a role in mRNA metabolism as a component of mRNP granules whose assembly is dependent on mRNA availability. TcXRNA granules colocalise with distinct RNP granules at the nuclear periphery, suggesting that the perinuclear region is a regulatory compartment in T. cruzi mRNA metabolism

Small-Subunit rRNA Processome Proteins Are Translationally Regulated during Differentiation of Trypanosoma cruzi

We used differential display to select genes differentially expressed during differentiation of epimastigotes into metacyclic trypomastigotes in the protozoan parasite Trypanosoma cruzi. One of the selected clones had a sequence similar to that of the small-subunit (SSU) processome protein Sof1p, which is involved in rRNA processing. The corresponding T. cruzi protein, TcSof1, displayed a nuclear localization and is downregulated during metacyclogenesis. Heterologous RNA interference assays showed that depletion of this protein impaired growth but did not affect progression through the cell cycle, suggesting that ribosome synthesis regulation and the cell cycle are uncoupled in this parasite. Quantitative PCR (qPCR) assays of several SSU processome-specific genes in T. cruzi also showed that most of them were regulated posttranscriptionally. This process involves the accumulation of mRNA in the polysome fraction of metacyclic trypomastigotes, where TcSof1 cannot be detected. Metacyclic trypomastigote polysomes were purified and separated by sucrose gradient sedimentation. Northern blot analysis of the sucrose gradient fractions showed the association of TcSof1 mRNA with polysomes, confirming the qPCR data. The results suggest that the mechanism of regulation involves the blocking of translation elongation and/or termination

Expression of non-acetylatable lysines 10 and 14 of histone H4 impairs transcription and replication in Trypanosoma cruzi

Submitted by Luciane Willcox ([email protected]) on 2016-10-13T19:10:55Z No. of bitstreams: 1 Expression of non-acetylatable lysines.pdf: 2595939 bytes, checksum: 7100c540b068524e05c9893932c2aa12 (MD5)Approved for entry into archive by Luciane Willcox ([email protected]) on 2016-10-13T19:17:21Z (GMT) No. of bitstreams: 1 Expression of non-acetylatable lysines.pdf: 2595939 bytes, checksum: 7100c540b068524e05c9893932c2aa12 (MD5)Made available in DSpace on 2016-10-13T19:17:21Z (GMT). No. of bitstreams: 1 Expression of non-acetylatable lysines.pdf: 2595939 bytes, checksum: 7100c540b068524e05c9893932c2aa12 (MD5) Previous issue date: 2015-11-19Fundac¸ ão de Amparo à Pesquisa do Estado de São Paulo—FAPESP (2011/51973-3) and by Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq (445655/2014-3 and 477143/2011-3 from the Instituto Nacional de Ciência e Tecnologia de Vacinas)Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia. São Paulo, SP, Brasil.The histone H4 from Trypanosomatids diverged from other eukaryotes in the N-terminus, a region that undergoes post-translation modifications involved in the control of gene expression, DNA replication, and chromatin assembly. Nonetheless, the N-terminus of Trypanosoma cruzi histone H4 is mainly acetylated at lysine 4. The lysines 10 and 14 are also acetylated, although at less extent, increasing during the S-phase or after DNA damage, which suggests a regulatory function. Here, we investigated the roles of these acetylations by expressing non-acetylated forms of histone H4 in T. cruzi. We found that histone H4 containing arginines at positions 10 or 14, to prevent acetylation were transported to the nucleus and inserted into the chromatin. However, their presence, even at low levels, interfered with DNA replication and transcription, causing a significant growth arrest of the cells. The absence of acetylation also increased the amount of soluble endogenous histones H3 and H4 and affected the interaction with Asf1, a histone chaperone. Therefore, acetylation of lysines 10 and 14 of the histone H4 in trypanosomes could be required for chromatin assembly and/or remodeling required for transcription and replication

DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions

Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. in mammals, DNA polymerase beta (Pol beta) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpol beta) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpol beta showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpol beta overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpol beta was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpol beta during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle. (C) 2012 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)PRONEXFundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Howard Hughes Medical InstituteUniv Fed Minas Gerais, Dept Biochem & Immunol, ICB UFMG, Inst Biol Sci, BR-31270901 Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniv Fed Rio de Janeiro, Hertha Meyer Cellular Ultra Struct Lab, Inst Biophys Carlos Chagas Filho, BR-21941 Rio de Janeiro, BrazilSuper Learning & Dev Ctr CESED, Sch Med Sci, Campina Grande, Paraiba, BrazilCNRS, Inst Pharmacol & Struct Biol, UMR5089, Toulouse, FranceUniv Toulouse, Toulouse, FranceUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilCNPq: MCT/CNPq/MS-SCTIE-DECIT 25/2006-Estudo de Doencas NegligenciadasWeb of Scienc